小鼠胚胎成纤维细胞-丝裂霉素处理Cell Specification
Mouse Embryonic Fibroblasts (MEF) are used to support the growth of mouse and human
pluripotent stem cells [1]. MEF not only provide a substrate for pluripotent stem cells to grow
on, but also secrete critical growth factors to maintain stem cell pluripotency. MEF are isolated
from mouse embryos and used at early passages [2]. To serve as feeder cells, MEF must be
treated with mitomycin C or by irradiation to prevent cell proliferation. The treated cells can also
be used to generate conditioned medium for feeder-free culture of pluripotent stem cells.
MEF-mt from ScienCell Research Laboratories are isolated from embryonic day 13 CD1 mouse
embryos. These cells have been treated with mitomycin C to prevent further cell division. They
are cryopreserved at passage 3 and delivered frozen. Each vial contains 1 x 106 cells in 1 ml
volume. MEF-mt are characterized by immunofluorescence with antibody specific to fibronectin.
MEF-mt are negative for mycoplasma, bacteria, yeast, and fungi. MEF are guaranteed to further
culture under the conditions provided by ScienCell Research Laboratories.
Recommended Medium
It is recommended to use DMEM (Cat. #09221) supplemented with 10% fetal bovine serum
(FBS, Cat. #0010, 0025, 0500) for culturing MEF-mt in vitro.
Product Use
小鼠胚胎成纤维细胞-丝裂霉素处理MEF-mt are used as feeder layer in mouse and human pluripotent stem cell culture. They are for
research use only. They are not approved for human or animal use, or for application in in vitro
diagnostic procedures.
Storage
Upon receiving, directly and immediately transfer the cells from dry ice to liquid nitrogen and
keep the cells in liquid nitrogen until they are needed for experiments.
Shipping
Dry ice.
References
[1] Bradley A. (1987) “Production and analysis of chimaeras”. In Robertson EJ, Teratocarcinomas and Embryonic
Stem Cells: A Practical Approach (pp 113-51). Oxford: IRL Press.
[2] Nagy A, Gertsenstein M, Vintersten K, Behringer R. (2006) “Preparing Mouse Embryo Fibroblasts”. Cold
Spring Harbor Protocols. pdb.prot 4398.
Instructions for culturing cells
Caution: Cryopreserved cells are very delicate. Thaw the vial in a 37o
C water bath
and return them to culture as quickly as possible with minimal handling!
Initiating the culture:
1. Prepare 0.1% gelatin (Cat. #0423) coated culture vessel. Use enough volume of gelatin to
cover the entire culture surface. Leave the vessel in a 37o
C incubator for 1 hour.
2. Prepare complete medium. Decontaminate the external surfaces of medium bottle and
medium supplement tubes with 70% ethanol and transfer them to a sterile field.
Aseptically transfer 10% FBS (Cat. #0010, 0025 or 0500) to DMEM (Cat. #09221).
3. Completely aspirate gelatin from the coated vessel. It is not necessary to rinse the vessel.
Add complete medium to cover the culture surface. Leave the vessel in the sterile field
and proceed to thaw the cryopreserved cells.
4. Place the frozen vial in a 37o
小鼠胚胎成纤维细胞-丝裂霉素处理C water bath. Hold and rotate the vial gently until the
contents completely thaw. Promptly remove the vial from the water bath, wipe it down
with 70% ethanol, and transfer it to the sterile field.
5. Carefully remove the cap without touching the interior threads. Gently resuspend and
dispense the contents of the vial into a 15 ml conical centrifuge tube. Add appropriate
volume of complete medium to the tube, gently mix well and plate cells into the
equilibrated, gelatin-coated culture vessel at required plating density.
Note: Dilution and centrifugation of cells after thawing are not recommended since these
actions are more harmful to the cells than the effect of residual DMSO in the culture. It is
also important that cells are plated in gelatin coated culture vessels to promote cell
attachment.
6. Replace the cap or lid of the culture vessel and gently rock the vessel to distribute the
cells evenly. Loosen cap, if necessary, to allow gas exchange.
7. Return the culture vessel to the incubator.
8. For best results, do not disturb the culture for at least 16 hours after the culture has been
initiated. Refresh culture medium the next day to remove residual DMSO and unattached
cells. Cells are then ready for experiments as feeder layer.
MEF mt are not recommended to be subcultured as mitomycin C treatment prevents
further cell proliferation.
小鼠胚胎成纤维细胞-丝裂霉素处理Caution: Handling animal derived products is potentially biohaza dous. Always wear gloves
and safety glasses when working with these materials. Never mouth pipette. We recommend
following the universal procedures for handling products of animal origin as the minimum
precaution against contamination [1].
[1]. Grizzle WE, Polt S. (1988) “Guidelines to avoid personal contamination by infective agents in research
laboratories that use human tissues.” J Tissue Culture Methods. 11: 191-9.